Tissue microarrays increase the throughput of molecular analyses by simultaneously arraying proteins, nucleic acids, and other biomolecules. The tissue microarrays allow for the study of protein expression, antigen localization and molecular profiling of tissues samples. These tissue microarrays typically include cylindrical biopsies of human or animal tissue taken from already fixed paraffin-embedded tissue blocks or fresh frozen tissues. The cylindrical biopsies are the positioned into a tissue recipient microarray block, which can then be histologically prepared to produce sections for subsequent study. Tissue microarrays can provide a great number of tissues to be screened on a single slide and allow for rapid, large scale assessment of tissues compared to conventional histological techniques.
In U.S. Pat. No. 6,103,518 to Leighton, incorporated herein by reference, Leighton describes a microarrayer instrument for automatically creating tissue microarrays. An example of this microarrayer is shown in FIG. 1. In the illustrated embodiment, a smaller recipient punch/needle 1 is used for making holes in a recipient block 4. A larger donor punch/needle 2, having an inner diameter corresponding to the outer diameter of the recipient punch 1, is used to obtain tissue core samples from the donor block 19. The donor punch 2 then embeds the tissue core samples into holes in the recipient block 4 formed by recipient punch 1. Each punch 1, 2 is provided with its own stylet/plunger for clearing material within the punch 1, 2, and each stylet has an outer diameter approximating that of the inner diameter of its respective punch 1, 2. At its greatest extension, the tip of the stylet slides past the tip of the punch 1, 2.
Blocks with recesses 24, 25 and clips are used to hold the respective punches 1, 2 on a pivot arm 26. The pivot arm 26 is pivotably mounted on a vertical carriage or slide 7 by a pivot bearing 3. The slide 7 moves vertically (z-axis) on a rail 28, which moves front to back (y-axis) on a horizontal slide 8 controlled by a drive 10. The horizontal slide 8 moves laterally (x-axis) on another slide 9 controlled by another drive 11. The slide 9 is affixed to a base plate 6.
Spacer bars 13, 14 are affixed to pivot arm 26, and each spacer bar 13, 14 is provided with an adjustment screw 15. Each screw 15 contacts an opposite side of the same stop 12, which is fixed with respect to the slide 7, thereby limiting the pivot movement and defining when the pivot arm is in either the first or second position. With the adjustment screws 5, the exact end of the travel can be set precisely. At one end of travel, the donor punch 2 is positioned in registry over a specific hole in the recipient block, and at the other end of the travel, the recipient punch 1 is precisely over the same location, assuming that the x- and y-axis drivers 10 and 11 have not been actuated.
Magnets 23 are fixed to the base 6 and hold a ferromagnetic plate 22 against the base plate 6 and against curbs or stops 21, 27. The ferromagnetic plate 22 is part of a recipient block holder 5, which holds recipient block 4. The holder 5, which includes the plate 22 and contains the block 4, can alternatively be easily removed from and reinserted to the same position on the base 6, or firmly held in place. The block 4 is formed from paraffin, or a like material, and individual recipient holes or an entire grid pattern of recipient holes may be cored into the recipient block 4 prior to harvesting samples from the donor block 4. The donor tissue block 19 rests on a removable bridge 20 that can be seated within stops 21, 27 or straddling the recipient block holder 22.
A problem associated with the creation of tissue microarrays using the apparatus and method described above is the removal of frozen embedding material from the recipient block using the recipient punch. As a needle of the punch is inserted into the embedding material, the embedding material immediately adjacent the needle is warmed by the needle and turns to liquid, and this liquefied embedding material adjacent the needle and frozen core of embedding material reduces the friction between the needle and frozen core. During an attempt to remove the frozen core of embedded material from the recipient block by withdrawing the needle, if insufficient friction exists between the frozen core of embedded material and the needle, the frozen core will slide relative the needle, and the frozen core will not be removed from the recipient block.
Another problem associated with the creation of tissue microarrays is the difficulty in creating cores in the recipient block that have a consistent depth. To solve this problem, the above-described microarrayer includes a depth stop kit that enables cores of an exact depth to be sampled from the donor block and the recipient block. This system allows paraffin-processed cores of an exact depth to be placed in holes having an identical depth. Since the paraffin-processed core is solid, the user of the instrument can easily determine when the core is fully inserted by detecting the resistance resulting from the solid bottom of the core contacting the bottom of the hole in the recipient block.
Paraffin-processed tissue cores, however, may not be the best option to demonstrate specific antigens. To solve this problem, whole frozen tissue cores are being used. However, the techniques used to control the size of the cores have problems when used with whole frozen tissues as the ability of an user to detect when a core fully inserted into a recipient block is compromised when the core is formed from whole frozen tissue. As the core is taken from the donor block, the material immediately adjacent the plunger may thaw slightly due to the temperature differential between the plunger and the frozen material. When thawing of the frozen material occurs, the user inserting the core into the recipient block does not detect exactly when the bottom of the core contacts the bottom of the hole in the recipient block because the thawed material flexes. Since the user cannot detect when the core is fully inserted, the core can be over-inserted into the hole in the recipient block, which results in the core being crushed. Since tissue is fragile and can be damaged by crushing, there is, therefore, a need for an improved tool and methodology for creating frozen tissue microarrays that reduces that incidence crushing of the tissue used to create the microarrays.